The hypothesis proposed in this application will produce significant breakthrough in the area of apoptotic neuron death. Apoptotic cells undergo marked cell volume decrease. Recent studies show that K+ efflux and subsequent cellular K+ depletion are likely prerequisites for the apoptotic shrinkage, caspase/endonuclease activation, and apoptosis. It has been well documented in cell volume regulation that K+ efflux is coupled with Cl- efflux to maintain electroneutrality. This proposal will test the hypothesis that Cl- homeostasis plays an important role in apoptosis either by affecting K' efflux or by influencing the cell volume regulation. We will study the Cl- hypothesis in vitro and in vivo. In vitro studies will use whole-cell and gramicidin-perforated patch recording, intracellular ion assay, and a variety of apoptosis assays in cultured cortical neurons; in vivo studies will be carried out in rats of transient cerebral ischemia. Three specific aims will be addressed in the proposed investigation. Aim 1. Cl- channel modulation and cellular Cl- loss induced by apoptotic insults. We hypothesize that apoptotic insults activate Cl- channels and stimulate Cl- efflux in neurons; the pro-apoptotic K+ efflux and cellular K' depletion may be facilitated by the Cl- channel up-modulation and enhanced Cl- efflux. We will examine the effects of apoptotic stimuli TNFa and ceramide on the outward rectifier chloride current (ORCC) and intracellular Cl- concentration/content. A comparison of the effects of blocking Cl- and K+ channels on intracellular Cl- and K+ will help characterize the relationship between the Cl- channel activation and reductions of intracellular Cl- and K+. Aim 2. Role of cell volume regulation in neuronal apoptosis. Information on the volume regulation in neurons is limited. We hypothesize that neurons undergoing apoptosis have enhanced regulatory volume decrease (RVD) and/or deficient regulatory volume increase (RVI). We will study the cell volume responses of cortical neurons to hyper- and hypoosmotic stresses under normal and apoptotic conditions as well as look at the effects of blocking Cl- and K+ movements on apoptotic volume decrease (AVD) and on RVD. Aim 3. Neuroprotective effects of Cl- blockers on neuronal apoptosis in vitro and in vivo. Blocking Cl- channels may not only attenuate CV, K+ efflux and cell shrinkage, but it is also favorable for retaining a negative membrane potential. We will study the potential inhibitory effects of blocking ORCC on cellular Cl- and K+ depletion, caspase activation, DNA fragmentation, and apoptotic death in cultured neurons. In vivo studies in rats will focus on evaluating the effect of Cl- channel blockers on the delayed apoptotic cell death and infarct volume induced by a transient cerebral ischemia. Information gained from this investigation will likely have significant impacts on the study of apoptosis and will lay the groundwork for a further investigation of the ionic mechanism of apoptosis. The study of neuroprotective effects of Cl- channel blockers will provide an opportunity for exploring a novel therapeutic approach for ischemic stroke and neurodegenerative diseases.